Name: two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification
Uploaded: 2021-03-31T13:30:00
Description: Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

This research was funded by Megaproject of Infectious Disease Control from Ministry of Health of China, grant number 2017ZX10302301-005 and Sino-Africa Joint Research Center, grant number SAJC201605.

Ethical statement 
Wuhan Institute of Virology (WHIOV) is among the labs and institutes approved by China CDC of Wuhan city to conduct research on SARS-CoV-2 and detect COVID-19 from clinical samples. Research on developing new diagnostic techniques for COVID-19 using clinical samples has also been approved by the ethical committee of Wuhan Institute of Virology (2020FCA001).
1. Sample processing workflow (Figure 1A)
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<ol>
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Few resources are available on how to develop RT-ddPCR assays for SARS-CoV-2 detection. Though not used in this article, standard samples with known copies may be used to develop and optimize assays. In this work however, SARS-CoV-2 samples grown in Vero-E6 cells were spiked in a background of human genomic RNA and used as standard samples to develop the assays. Proper primer and probe sequences are essential when developing assays. Since most preliminary work on SARS-CoV-2 RT-ddPCR used the China CDC primer and probes t...

Polymerase chain reaction (PCR), a well-recognized technique, has undergone several transformations since its advent to become a powerful technique capable of providing answers to nucleic acid research. These transformations have been a constant improvement of the old technique. These transformations can be summarized into three generations1. The first generation is conventional PCR that relies on gel electrophoresis to quantify and detect amplified targets. The second generation is quantitative real time PCR (qPCR) that can detect samples in real time and rely on a standard curve to directly quantify targets in a sample. The third generation, digital PCR (dPCR), can perform both detection, and absolute quantification of nucleic acid targets without the need of a standard curve. dPCR has also been improved further from reaction chambers being separated by the wells of a wall into emulsions of oil, water, and stabilizing chemicals within the same well as seen in droplet-based digital PCR2. In droplet digital PCR (ddPCR), a sample is partitioned into thousands of nanoliter-sized droplets containing individual targets that will later be quantified using Poisson statistics2,3,4. This technique gives ddPCR an edge in quantifying low abundant targets when compared to the other generations of PCR.
Recently, multiple applications have highlighted the superiority of ddPCR over the commonly used qPCR when detecting and quantifying low abundant targets1,5,6. SARS-CoV-2 is no exception to these applications7,8,9,10,11,12. Since the outbreak of SARS-CoV-2, scientists have been working on all fronts to come up with solutions on how to diagnose the virus and detect it efficiently. The current gold standard still remains to be qPCR13. However, RT-ddPCR has been shown to be more accurate in detecting low abundant SARS-CoV-2 targets from both environmental and clinical samples when compared to RT-qPCR7,8,9,10,11,12. Most of the SARS-CoV-2 ddPCR published works depend on simplex assays with the multiplex ones depending on commercial assays. Hence, more should be done to explain how to develop multiplex RT-dPCR assays for SARS-CoV-2 detection.
In a proper assay design, multiplexing can be used to save on cost, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. When multiplexing with ddPCR, one must take account of how many fluorophores can be detected in a particular system. Some ddPCR platforms can support up to three channels while others support only two channels. Hence, when multiplexing with two channels, one has to use different approaches, including higher order multiplexing to detect more than two targets14,15,16. In this work, a two color ddPCR detection system is used to show steps on how to develop different SARS-CoV-2 RT-ddPCR assays that can be adapted for different research applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32-channel fully automatic nucleic acid extractor Purifier 32</td>
			<td>Genfine Biotech</td>
			<td>FHT101-32</td>
			<td>Automated extractor for RNA</td>
		</tr>
		<tr>
			<td>AutoDG Oil for Probes</td>
			<td>BioRad</td>
			<td>12003017</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>BioRad</td>
			<td>12003185</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>BioRad</td>
			<td>1863024</td>
			<td>Making ddPCR assay mastermix</td>
		</tr>
		<tr>
			<td>DG32 AutoDG Cartridges</td>
			<td>BioRad</td>
			<td>1864108</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>Electronic thermostatic water bath pot</td>
			<td>Beijing Changfeng Instrument and Meter Company</td>
			<td>XMTD-8000</td>
			<td>Heat inactivation of samples</td>
		</tr>
		<tr>
			<td>FineMag Rapid Bead Virus DNA/RNA Extraction Kit</td>
			<td>Genfine Biotech</td>
			<td>FMY502T5</td>
			<td>Magnetic bead extraction of inactivated RNA samples</td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seals</td>
			<td>BioRad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips for the AutoDG</td>
			<td>BioRad</td>
			<td>1864120</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>Pipet Tip Waste Bins for the AutoDG</td>
			<td>BioRad</td>
			<td>1864125</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Mix (Perfect Real Time)</td>
			<td>TaKaRa</td>
			<td>RR036A</td>
			<td>cDNA generation</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>BioRad</td>
			<td>1814000</td>
			<td>Seal the droplet plate from AutoDG</td>
		</tr>
		<tr>
			<td>QuantaSoft 1.7 Software</td>
			<td>BioRad</td>
			<td>10026368</td>
			<td>Data acquisition and analysis</td>
		</tr>
		<tr>
			<td>QuantaSoft Analysis Pro 1.0</td>
			<td>BioRad</td>
			<td>N/A</td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>QX200 Automated Droplet Gererator (AutoDG)</td>
			<td>BioRad</td>
			<td>1864101</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>BioRad</td>
			<td>1864003</td>
			<td>Droplet reading and data acquisition</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td>Droplet target amplification (PCR) and cDNA generation</td>
		</tr>
	</tbody>
</table>

two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

In a proof-of-concept study, the multiplex assays analytical performance was tested on clinical and research samples19. The performance of the multiplex assays was superior to that of an RT-PCR19. Since low numbers of droplets may indicate a problem during droplet generation, in this article a cutoff of 10,000 droplets per well was set based on empirical data.
A good separation between positive and negative droplets with minimal rain interference...

Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

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